Microfluidic system incorporating light absorbing materials

ABSTRACT

Systems and methods for light based heating of light absorbing sources for modification of nucleic acids through fast thermal cycling of polymerase chain reaction are provided. The system includes a polymeric fluidic device comprising one or more reaction wells. A first light absorbing material is disposed on a first support to define a reaction well and first and second ports are coupled to the reaction wells. The first and second ports are configured to allow input of a fluidic sample into the reaction well. A lyophilized reagent is pre-loaded in the reaction well. A light source is configured to illuminate the first light absorbing material. A first portion of light illuminated onto the first light absorbing material is absorbed into the first light absorbing material and is configured to elevate the temperature of the first light absorbing material to heat the fluidic sample within the reaction well.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/643,494, filed on Mar. 15, 2018, and U.S. Provisional PatentApplication No. 62/652,861, filed on Apr. 4, 2018, the contents of bothof which are hereby incorporated by reference in their entirety for allpurposes.

The following regular U.S. patent applications (including this one) arebeing filed concurrently, and the entire disclosure of the otherapplications are incorporated by reference into this application for allpurposes:

The following regular U.S. patent applications (including this one) arebeing filed concurrently, and the entire disclosure of the otherapplications are incorporated by reference into this application for allpurposes:

Application No. 16/353,907, filed Mar. 14, 2019, entitled “Method andSystem for Performing Heat Assisted Biochemical Reactions”;

Application No. 16/353,926, filed Mar. 14, 2019, entitled “MicrofluidicSystem Incorporating Light Absorbing Materials”.

BACKGROUND OF THE INVENTION

Polymerase chain reaction (PCR), is an important technique in the fieldsof clinical laboratories, environmental science, forensic science andagricultural science. There is a need for rapid and accurate diagnosticsof nucleic acids. Fast/ultrafast PCR is desirable for applications suchas time-sensitive diagnosis of diseases, genetic disorders, andlaboratory experiments amongst other applications. Accordingly, anultrafast PCR system would be desirable for laboratory testing, andpoint of care testing that is robust, simple, easy to use andcharacterized by low power consumption.

SUMMARY OF THE INVENTION

In some aspects, a system is provided. The system can be utilized fornucleic acid modification. The system may comprise a transparent blockwhich may comprise one or more intrusions. The system may comprise alight absorbing material disposed within the one or more intrusions ofthe transparent block. Additionally, the system may comprise a reactionvessel removably positioned onto the intrusions of the transparentblock. The system may comprise a light source. The light source may beconfigured to be directed at the intrusions of the transparent blocksuch that light from the light source may generate heat within the lightabsorbing material subsequently heating the reaction vessel.

In some aspects, a system is provided. The system can be used fornucleic acid modification and may comprise a polymeric reaction vesselcomprising one or more wells. The system may comprise a light absorbingmaterial disposed within the one or more wells of the reaction vessel.The system may comprise a transparent block with intrusions to hold thereaction vessel. It may also comprise a light source. The light sourcemay be configured to be directed at the wells of the transparent blocksuch that light from the light source may generate heat within the lightabsorbing material and may heat the reaction vessel. The system may alsocomprise a sealing film disposed on the reaction vessel.

In some aspects, a system is provided. The system can be used fornucleic acid modification and may comprise a polymeric fluidic devicecomprising one or more reaction wells. The system may comprise a firstlight absorbing material disposed on a first support to define areaction well and a second light absorbing material disposed on a secondsupport opposite the first support. The first and second ports may becoupled to the reaction wells; wherein the first and second ports may beconfigured to allow input of a fluidic sample into the reaction well. Alyophilized reagent may be pre-loaded on the reaction well. The systemmay further comprise a light source configured to illuminate the firstlight absorbing material; wherein a first portion of light illuminatedonto the first light absorbing material may be absorbed into the firstlight absorbing material and a second portion of the light illuminatedonto the first light absorbing material may be transmitted through thefirst light absorbing material. The light transmitted through the firstlight absorbing material may illuminate the second light absorbingmaterial; wherein at least a portion of the transmitted lightilluminated onto the second light absorbing material may be absorbedinto the second light absorbing material. The absorbed light into thefirst light absorbing material and second light absorbing material maybe configured to uniformly elevate a temperature of the first lightabsorbing material and second light absorbing material which may lead toheating of the fluidic sample within the reaction wells.

In some embodiments, the system may further comprise a sealing filmdisposed on the reaction vessel.

In some embodiments, the transparent block material may comprisetransparent polymer, Polydimethylsiloxane (PDMS), glass,polymethylmethacrylate (PMMA), cyclic olefin copolymer (COC) and/orquartz.

In some embodiments, the shape of intrusions of the transparent blockmay be conical, hemispherical, pyramidal, rectangular, cylindrical,truncated, or dome-shaped. In some embodiments, an additional channel isplaced around one or more of the intrusions.

In some embodiments, the transparent block may comprise a fluidcirculation channel. In some embodiments, air/water/liquid flows throughthe circulation channel.

In some embodiments, the light absorbing material may comprise metallicthin film, non-metallic thin film, graphite, graphene, carbon nanotube,and/or paint. In some embodiments, the metallic thin film may comprise asingle or multi-layer metallic structure; the metallic structurecomprising one or more metals may be selected from the group comprisingof: gold (Au), silver (Ag), nickel (Ni), titanium (Ti), chromium (Cr),germanium (Ge), palladium (Pd), ruthenium (Ru), tungsten (W), iridium(Ir), or platinum (Pt).

In some embodiments, the reaction vessel may comprise wells. The shapeof the wells in the reaction vessel may be the same as the shape of theintrusions in the transparent block.

In some embodiments, the thickness of the reaction vessel may be lessthan 1 mm.

In some embodiments, the light source may be a light-emitting diode(LED), laser diode (LD), tungsten lamp, fluorescent lamp, halogen lamp,mercury lamp, xenon lamp, metal halide lamp, or combination thereof.

In some embodiments, the reaction vessel may be a PCR tube, a PCR plate,or a PCR strip.

In some embodiments, the system may further comprise one or more lightsources. The number of light sources may be equal to the number ofintrusions in the transparent block.

In some embodiments, an emission wavelength of the light source may notoverlap with an excitation wavelength of a fluorescent dye used forreal-time detection of nucleic acids.

In some embodiments, the sealing film further may comprise a lightabsorbing layer. In some embodiments, the sealing film may be colored.

In some embodiments, the system may further comprise one or moretemperature sensors configured to monitor the temperature.

In some embodiments, the system may further comprise one or moreexcitation LEDs configured for excitation of a fluorescent dye.

In some embodiments, the system may further comprise one or more opticalfilters for the excitation LED.

In some embodiments, the system may further comprise two or moreexcitation LEDs. Each of the excitation LEDs can have a differentwavelength to excite two or more fluorescent dyes.

In some embodiments, a high refractive index material may be disposedoutside of the transparent heat block for internal reflection of lightfrom an excitation LED.

In some embodiments, the system may further comprise a CMOS sensor, CCDsensor, photodiode or spectrophotometer.

In some embodiments, the system may further comprise a first filter foremission of a fluorescent dye and a second filter for elimination oflight from the light source.

In some embodiments, the second filter may be a Distributed Braggreflector (DBR) with high reflectivity over the emission wavelength ofthe light source.

In some embodiments the system may further comprise an embedded lens tofocus an emissive light from a fluorescent dye. In some embodiments, thesystem may further comprise a lens disposed between the light source andthe transparent block. The lens may be configured to focus emissivelight from a light source. Alternatively, the lens may be configured tofocus emissive light from a fluorescent dye.

In some embodiments, the photodiode may be an IR sensitivity suppressedtype.

In some embodiments, lyophilized PCR reagents may be pre-loaded in thereaction vessel.

In some embodiments, the sealing film may comprise a sealing plate withor without a light absorbing material on the surface of the plate.

In some embodiments, the system may further comprise a processor, theprocessor may be coupled to the light source and may be configured withinstructions to heat the reaction vessel with the light source.

In some embodiments the system may further comprise a processor. Theprocessor may be coupled to the circulation channel. The processor maybe configured with instructions to cool the reaction vessel.

In some embodiments, the light source may be pulsed for the photothermalheating of the light absorbing material.

In some embodiments, a duty cycle of pulsed operation may be from 1% to100%.

In some embodiments, the light source and the excitation source may bepulsed in an alternating manner.

In some embodiments, a signal for detection of nucleic acidsmodification may be detected during an off cycle of pulsed operation ofthe light source.

In some embodiments, the light absorbing material further may comprise apassivation layer to prevent PCR reaction inhibition within the thermalcycling chamber. In some embodiments, the passivation layer may comprisean oxide thin film or a thin polymeric layer.

In some embodiments, one or more intrusions in the wells may comprise2-D or 3-D microstructures or nanostructures in the form of a pillararray, 1D or 2D grating, photonic crystal, or hemi-sphere.

In some embodiments, reagents may be lyophilized. The lyophilizedreagent may comprise a primer set for PCR. In some embodiments, thelyophilized reagent may comprise PCR reagent and primer set. In someembodiments, the lyophilized regent further may comprise a stabilizingreagent.

In some embodiments, the stabilizing reagent may comprise paraffin waxor hydrogel.

In some embodiments, the system may further comprise a fluidic valvebetween wells. In some embodiments, the fluidic valve may be operated byan external controller. The system may further include a lens disposedbetween the light source and the polymeric fluidic device.

In some embodiments, the polymeric fluidic device includes a fluidcirculation channel. As an example, air, water, and /or liquid can flowthrough the circulation channel.

In some embodiments, the system may comprise a sample preparationmodule. The sample preparation module may comprise multiplecompartments, and a cartridge; and a microfluidic PCR device. Themicrofluidic PCR device may comprise photonic PCR wells.

In some embodiments, the sample preparation module may further comprisea lysis system for photo-thermal lysis of cells.

In some embodiments, the sample preparation module may comprise one ormore filters in a chamber. In some embodiments, the sample preparationmodule may comprise a first filter and a second filter wherein the firstand second filter have different pore sizes. In some embodiments, afirst filter may be used to remove large debris, crystals and/or largecells from a sample.

In some embodiments, a second filter may trap cells of interest based onthe size of cells. In some embodiments, nucleic acids may be extractedfrom the cells trapped on the second filter. In some embodiments, thesecond filter may comprise a layer of light absorbing material. In someembodiments, a chamber below the second filter may comprise a layer oflight absorbing material.

In some embodiments, the sample preparation module may comprisecompartments further comprising electrodes placed around the compartmentto change the pH of the solution by applying voltage.

In some embodiments, the sample preparation module may comprise a wastechamber. In some embodiments, the waste chamber may comprise absorbingporous paper, fabric or sponges to prevent re-flux of fluid.

In some embodiments, the sample preparation module may further comprisea microfluidic device comprising cartridges with wells.

In some embodiments, wells in the cartridges may be pre-loaded with aprimer and probe set for the detection of target nucleic acids.

In some embodiments, a well in the cartridges may comprise a set ofprimers and probes to detect one target nucleic acid.

In some embodiments, a well in the cartridge may comprise a set ofprimers and probes to detect multiple target nucleic acids.

In some embodiments, the system may comprise a light absorbing material,wherein the light absorbing material may comprise one or more openareas. In some embodiments, the open area may form 1% to 90% of thelight absorbing material.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “figure” and “FIG.” herein), of which:

FIG. 1 is a schematic depicting an example system, in accordance withsome embodiments.

FIG. 2A is a schematic of the system, which may be used for modificationof nucleic acids, in accordance with some embodiments.

FIG. 2B is an illustration of shapes of wells or intrusions in a system,in accordance with some embodiments.

FIG. 2C and 2D are schematics of sealing films to be used with a system,in accordance with some embodiments.

FIG. 2E is a schematic of the placement of an embedded lens above areaction vessel, in accordance with some embodiments.

FIG. 3 is an illustration of the system with an inlet channel and anoutlet, in accordance with some embodiments.

FIG. 4 is a schematic of a system with a fluid channel, in accordancewith some embodiments.

FIG. 5 is a schematic of a system with a detailed view of the opticalsignal measurement apparatus, in accordance with some embodiments.

FIG. 6 is an exploded view of a schematic of a system, in accordancewith some embodiments.

FIG. 7 is a schematic and dimensions of a reaction vessel system, inaccordance with some embodiments.

FIG. 8 is a schematic depiction of the mechanism of loading a system, inaccordance with some embodiments.

FIGS. 9A-9C are schematics of different forms of light absorbingmaterial, in accordance with some embodiments.

FIG. 10 is a schematic depiction of continuous and pulsed operation ofthe light source, in accordance with some embodiments.

FIG. 11A is a schematic depiction of pulsed operation of the lightsource and the excitation source in conjunction with signal measurement,in accordance with some embodiments.

FIG. 11B is a schematic depiction of pulsed operation of the lightsource and continuous operation of the excitation source in conjunctionwith signal measurement, in accordance with some embodiments.

FIG. 12 is an exploded view of the sample preparation module with PCRreaction wells, in accordance with some embodiments.

FIGS. 13A-13E are a schematic depiction of the sample preparation modulein use, in accordance with some embodiments.

FIG. 14 is a schematic depiction of the sample preparation module withPCR reaction wells, in accordance with some embodiments.

FIG. 15 shows a non-limiting example of a digital processing device; inthis case, a device with one or more CPUs, a memory, a communicationinterface, and a display.

FIG. 16 shows a flowchart of a method for modifying nucleic acids anddetermining target nucleic acids, in accordance with some embodiments.

FIG. 17 shows a schematic depicting an example of using the methods andsystems for detection of a target cell.

FIGS. 18A-18C show representative end-point data for detection ofvarious gene sequences.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

In the following description, various aspects of the invention will bedescribed. For the purposes of explanation, specific details are setforth in order to provide a thorough understanding of the invention. Itwill be apparent to one skilled in the art that there are otherembodiments of the invention that differ in details without affectingthe essential nature thereof. Therefore the invention is not limited bythat which is illustrated in the figure and described in thespecification, but only as indicated in the accompanying claims, withthe proper scope determined only by the broadest interpretation of theclaims.

A better understanding of the features and advantages of the presentdisclosure will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of embodiments of the present disclosure are utilized, andthe accompanying drawings.

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. As used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. Any referenceto “or” herein is intended to encompass “and/or” unless otherwisestated.

The term “sample” us used herein may generally refer to a biologicalsample of a subject. The sample may be a tissue sample, such as abiopsy, core biopsy, needle aspirate, or fine needle aspirate. Thesample may be a fluid sample, such as a blood sample, urine sample, orsaliva sample. The sample may be a skin sample, a cheek swab. The samplemay be a plasma or serum sample.

Nucleic acids may be isolated from one or more samples. As used herein,the term “nucleic acid” generally refers to a polymeric form ofnucleotides of any length, either deoxyribonucleotides (dNTPs) orribonucleotides (rNTPs), or analogs thereof. Non-limiting examples ofnucleic acids include DNA, RNA, coding or non-coding regions of a geneor gene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A nucleic acid may compriseone or more modified nucleotides, such as methylated nucleotides andnucleotide analogs.

The term “nucleic acid modification” may generally refer tomodifications made to one or more nucleic acids. Modifications mayinclude but are not limited to amplification, denaturation, elongation,primer extension reactions, nucleotide analog addition, etc.

As used herein, the term “reagents” generally refers to a compositioncomprising reaction mixtures necessary to complete nucleic acidmodification (e.g., DNA amplification, RNA amplification), withnon-limiting examples of such reagents that include primer sets havingspecificity for target RNA or target DNA, DNA produced from reversetranscription of RNA, a DNA polymerase, a reverse transcriptase (e.g.,for reverse transcription of RNA), suitable buffers (includingzwitterionic buffers), co-factors (e.g., divalent and monovalentcations), dNTPs, and other enzymes (e.g., uracil-DNA glycosylase (UNG)),etc.). Reagents may also comprise reporter agents or fluorescent dyesfor incorporation in to an amplified product. In some cases, reagentscan also comprise one or more reporter agents. Reagents may belyophilized, stabilized or in a solution. Stabilization of the reagentsmay be performed using hydrogel or paraffin wax. Other methods ofstabilizing, lyophilizing such reagents known to one of ordinary skillin the art may be used.

The term “polymerase,” as used herein, generally refers to any enzymecapable of catalyzing a polymerization reaction. Polymerases may be usedextend primers with the incorporation of nucleotides or nucleotideanalogs. Examples of polymerases include, without limitation, a nucleicacid polymerase. The polymerase can be naturally occurring orsynthesized. In some cases, a polymerase has relatively highprocessivity. An example polymerase is a Φ29 polymerase or a derivativethereof. Examples of polymerases include a DNA polymerase, an RNApolymerase, a thermostable polymerase, a wild-type polymerase, amodified polymerase, E. coli DNA polymerase I, T7 DNA polymerase,bacteriophage T4 DNA polymerase Φ29 (phi29) DNA polymerase, Taqpolymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pwopolymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase,LA-Taq polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tnepolymerase, Tma polymerase, Tea polymerase, Tih polymerase, Tfipolymerase, Platinum Taq polymerases, Tbr polymerase, Tfl polymerase,Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase,Bst polymerase, Sac polymerase, Klenow fragment, polymerase with 3′ to5′ exonuclease activity, and variants, modified products and derivativesthereof.

As used herein, the terms “amplifying” and “amplification” are usedinterchangeably and generally refer to generating one or more copies or“amplified product” of a nucleic acid. Non-limiting examples of nucleicacid amplification methods include reverse transcription, primerextension, polymerase chain reaction, ligase chain reaction,helicase-dependent amplification, asymmetric amplification, rollingcircle amplification, and multiple displacement amplification (MDA). Incases where DNA is amplified, various DNA amplification methods may beemployed. Non-limiting examples of DNA amplification methods includepolymerase chain reaction (PCR), variants of PCR (e.g., real-time PCR,allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsionPCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR,inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR,nested PCR, overlap-extension PCR, thermal asymmetric interlaced PCR,touchdown PCR), and ligase chain reaction (LCR). Amplification may beused to incorporate nucleotides and/or nucleotide analogs in to agrowing chain of nucleic acids. PCR may be employed with thermal cyclingor isothermally (i.e., isothermal PCR). Reporter agents such asfluorescent dyes may be used to identify target nucleic acids.

Reporter agents may be linked with nucleic acids, including amplifiedproducts, by covalent or non-covalent means. Non-limiting examples ofnon-covalent means include ionic interactions, Van der Waals forces,hydrophobic interactions, hydrogen bonding, and combinations thereof. Insome embodiments, reporter agents may bind to initial reactants andchanges in reporter agent levels may be used to detect amplifiedproduct. In some embodiments, reporter agents may only be detectable (ornon-detectable) as nucleic acid amplification progresses. In someembodiments, an optically-active dye (e.g., a fluorescent dye) may beused as a reporter agent. Non-limiting examples of dyes include SYBRgreen, EvaGreen, LCGreen, SYBR blue, DAPI, propidium iodine, SYBR gold,ethidium bromide, acridines, proflavine, acridine orange, acriflavine,fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D,chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin,phenanthridines and acridines, ethidium bromide, propidium iodide,hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidiummonoazide, and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI,acridine orange, 7-AAD, actinomycin D, LDS751, hydroxystilbamidine,SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3,TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3,BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1,YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBRGreen I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45(blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25(green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59,-61, -62, -60, -63 (red), fluorescein, fluorescein isothiocyanate(FITC), tetramethyl rhodamine isothiocyanate (TRITC), rhodamine,tetramethyl rhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5,Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), Sybr Green I, SybrGreen II, Sybr Gold, CellTracker Green, 7-AAD, ethidium homodimer I,ethidium homodimer II, ethidium homodimer III, ethidium bromide,umbelliferone, eosin, green fluorescent protein, erythrosin, coumarin,methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow,cascade blue, dichlorotriazinylamine fluorescein, dansyl chloride,fluorescent lanthanide complexes such as those including europium andterbium, carboxy tetrachloro fluorescein, 5 and/or 6-carboxy fluorescein(FAM), 5- (or 6-) iodoacetamidofluorescein, 5-{[2(and3)-5-(Acetylmercapto)-succinyl]amino} fluorescein (SAMSA-fluorescein),lissamine rhodamine B sulfonyl chloride, 5 and/or 6 carboxy rhodamine(ROX), 7-amino-methyl-coumarin, 7-Amino-4-methylcoumarin-3-acetic acid(AMCA), BODIPY fluorophores, 8-methoxypyrene-1,3,6-trisulfonic acidtrisodium salt, 3,6-Disulfonate-4-amino-naphthalimide,phycobiliproteins, AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568,594, 610, 633, 635, 647, 660, 680, 700, 750, and 790 dyes, DyLight 350,405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes, or otherfluorophores.

The present disclosure relates to methods and systems for nucleic acidmodification and detection. The systems and methods may be used todetect multiple target nucleic acids samples and sequences. The methodsand systems may be used as point of care testing devices for detectionof infectious diseases, genetic abnormalities amongst other uses.

An example system for amplifying a target nucleic acid according tomethods described herein is depicted in FIG. 1. The system comprises acomputer 105, also referred to as a processor, that may serve as part ofboth the input and output modules. A user enters a sample/reactionvessel 103 comprising a reaction mixture ready for nucleic acidmodification into the thermocycler system 101. In some cases,sample/reaction vessel 103 may be a sample preparation module thatprepares the sample and loads the sample into a reaction vessel. Thethermocycler system 101 may be attached to an optical signal measurementapparatus 102. The input and output module 109 comprises processor 105and associated input devices 106 (e.g., tablets, keyboard, mouse, etc.)that can receive the user's request to amplify a target nucleic acid inthe reaction mixture. The processor 105 may use electronic display 107to send requests for modification of target sample preparation. Theinput and output module 109 communicates the user's request to thesample module 100 and nucleic acid modification commences in thethermocycler system 101. As modification proceeds, the optical signalmeasurement apparatus 102 of the sample module detects the amplifiedproduct. Information (e.g., raw data obtained by the detector) regardingthe amplified product is transmitted from the optical signal measurementapparatus 102 back to the processor 105, which also serves as acomponent of the input and output module 109. The processor 105 receivesthe information from the sample module 104 performs any additionalmanipulations to the information, and then generates results containingthe processed information. Results may be in the form of a report. Oncethe results are generated, the processor 105 then transmits the reportto its end recipient over a computer network (e.g., an intranet, theinternet) via computer network interface 108, in various forms.

FIG. 2A illustrates an example of the sample module 100. In someembodiments, a system or a sample module may include a transparent block110. Transparent block may be a conventional PCR block. Transparentblock may comprise transparent polymers, polydimethysiloxane (PDMS),glass, polymethylmethacrylate (PMMA), cyclic olefin copolymer (COC) andor quartz. The transparent block 110 may have one or more intrusions orwells 112 to removably place a reaction vessel 130 illustrated in FIG.4. Intrusions 112 in the transparent block may be conical,hemispherical, pyramidal, rectangular, cylindrical, truncated ordome-shaped. Some examples of the shapes of the intrusions in thetransparent block have been shown in FIG. 2B. The intrusions may be theshape of a conventional PCR tube, strip or plate. The number ofintrusions in a transparent block may be at least 8, 12, 14, 24, 36, 48,96, 100 or 384 wells. The distance between each intrusion may be thesame as the distance between the wells of a conventional PCR tube orplate.

Reaction vessels 130 as illustrated in FIG. 4 may include conventionalPCR tubes, strips or plates. Reaction vessels may be conical,hemispherical, pyramidal, rectangular, cylindrical, truncated ordome-shaped. Some examples of the shapes of wells in reaction vesselshave been shown in FIG. 2B. Reaction vessels may comprise thermoplasticpolymers including polystyrene, polypropylene, poly (methylmethacrylate), cyclic olefin copolymer, polycarbonate and/or theirderivatives. In some cases, the thickness of the reaction vessel may be0.01 mm to 4 mm. In some cases, the thickness of the reaction vessel maybe at least 0.01 mm. In some cases, the thickness of the reaction vesselmay be at most 4 mm. In some cases, the thickness of the reaction vesselmay be 4 mm to 3 mm, 4 mm to 2 mm, 4 mm to 1 mm, 4 mm to 0.5 mm, 4 mm to0.1 mm, 4 mm to 0.05 mm, 4 mm to 0.01 mm, 3 mm to 2 mm, 3 mm to 1 mm, 3mm to 0.5 mm, 3 mm to 0.1 mm, 3 mm to 0.05 mm, 3 mm to 0.01 mm, 2 mm to1 mm, 2 mm to 0.5 mm, 2 mm to 0.1 mm, 2 mm to 0.05 mm, 2 mm to 0.01 mm,1 mm to 0.5 mm, 1 mm to 0.1 mm, 1 mm to 0.05 mm, 1 mm to 0.01 mm, 0.5 mmto 0.1 mm, 0.5 mm to 0.05 mm, 0.5 mm to 0.01 mm, 0.1 mm to 0.05 mm, 0.1mm to 0.01 mm, or 0.05 mm to 0.01 mm. In some cases, the thickness ofthe reaction vessel may be less than 4 mm, 3 mm, 2 mm, 1 mm, 0.5 mm, 0.1mm, 0.05 mm, or 0.01 mm.

In some cases, the transparent block 110 may be used as a reactionvessel as is shown in FIG. 3. Transparent block 110 may compriseintrusions which may be used as reaction wells 131, 132, 133, etc. Insuch cases, the system may be part of a microfluidic system whereinsample input and output may be performed using ports. The transparentblock 110 may comprise one or more inlet ports 310 for the loading ofsamples and reagents as shown in FIG. 3. In some cases, the transparentblock may comprise one or more outlet ports 312.

As shown in FIG. 2A, the reaction vessel may comprise one or more lightabsorbing layers 120. In some cases, the transparent block with reactionwells (as shown in FIG. 3) may comprise a light absorbing layer 120. Insome cases, the light absorbing material 120 may be in direct contactwith reagents 180. In some embodiments, the light absorbing material maynot be in direct contact with the reagents. In some cases, the lightabsorbing material may have a passivation layer (not shown here) toprevent inhibition of enzymes. In some cases, one or more wells in areaction vessel may comprise 2-D or 3-D microstructures ornanostructures in the form of one or more pillar arrays, 1D or 2Dgrating structures, photonic crystals or hemispheres.

The light absorbing layer 120 may be in the shape of the reactionvessel. In some cases, the reaction vessel may be covered by the lightabsorbing layer. In some cases, a light absorbing layer may be depositedon the surface of the well. In some cases, the thickness of the lightabsorbing layer may be 1 nm to 1 mm. In some cases, the thickness of thelight absorbing layer may be at least 1 nm. In some cases, the thicknessof the light absorbing layer may be at most 1 mm. In some cases, thethickness of the light absorbing layer may be 1 nm to 50 nm, 1 nm to 100nm, 1 nm to 500 nm, 1 nm to 1,000 nm, 1 nm to 0.01 mm, 1 nm to 0.05 mm,1 nm to 0.1 mm, 1 nm to 0.5 mm, 1 nm to 1 mm, 50 nm to 100 nm, 50 nm to500 nm, 50 nm to 1,000 nm, 50 nm to 0.01 mm, 50 nm to 0.05 mm, 50 nm to0.1 mm, 50 nm to 0.5 mm, 50 nm to 1 mm, 100 nm to 500 nm, 100 nm to1,000 nm, 100 nm to 0.01 mm, 100 nm to 0.05 nm, 100 nm to 0.1 mm, 100 nmto 0.5 mm, 100 nm to 1 mm, 500 nm to 1,000 nm, 500 nm to 0.01 mm, 500 nmto 0.05 mm, 500 nm to 0.1 mm, 500 nm to 0.5 mm, 500 nm to 1 mm, 1,000 nmto 0.01 mm, 1,000 nm to 0.05 mm, 1,000 nm to 0.1 mm, 1,000 nm to 0.5 mm,1,000 nm to 1 mm, 0.01 mm to 0.05 mm, 0.01 mm to 0.1 mm, 0.01 mm to 0.5mm, 0.01 mm to 1 mm, 0.05 mm to 0.1 mm, 0.05 mm to 0.5 mm, 0.05 mm to 1mm, 0.1 mm to 0.5 mm, 0.1 mm to 1 mm, or 0.5 mm to 1 mm. In some cases,the thickness of the light absorbing layer may be 1 nm, 50 nm, 100 nm,500 nm, 1,000 nm, 0.01 mm, 0.05 mm, 0.1 mm, 0.5 mm, or 1 mm. Reactionvessels may comprise a lower light absorbing layer 120 and an upperabsorbing layer 121. The upper light absorbing layer may be a part ofthe sealing film.

Light absorbing layer 120 may comprise a layer of metals. Non-limitingexamples of metals that may be used are gold (Au), silver (Ag), nickel(Ni), titanium (Ti), chromium (Cr), germanium (Ge), palladium (Pd),ruthenium (Ru), tungsten (W), iridium (Ir), or platinum (Pt). In somecases, the light absorbing layer is an alloy. Light absorbing materialmay be a carbon base, non-limiting examples of which include carbonnanotubes, graphite, graphene and/or graphene oxide. Light absorbinglayer may comprise of paints such as acrylic paints. Light absorbinglayer may comprise a mixture of a metal, metal alloy, carbon base orpaint. In some cases, more than one layer of light absorbing materialsmay be used. One light absorbing layer may absorb light from a lightsource and transmit light which is not absorbed at first light absorbinglayer to the second light absorbing layer to generate heat. Lightabsorbing materials used may be thin to maintain high heating andcooling rates.

A reaction vessel may be sealed with a sealing film 150 as discussedmore fully with respect to FIG. 4. The sealing film may compriseadhesives. In some cases, the sealing film may comprise a lightabsorbing layer 152. The light absorbing layer in the sealing film mayhelp in maintaining a uniform temperature in the reaction vessel. Asealing film may comprise aluminum, polyolefin, polypropylene,polyester, polycarbonate, polystyrene and other commercially usedsealing film materials. A sealing film may be a conventional PCR platesealing film. In some cases, a sealing film may be colored to absorblight. A sealing film may comprise multiple layers as shown in FIGS.2C-2D. The sealing film may have a layer of adhesive material 151. Inaddition to the adhesive material 151, the sealing film may have a layerof light absorbing material and/or a colored film 152 as shown in FIG.2C. In some cases, in addition to the adhesive material, the sealingfilm may comprise a layer of light absorbing material 152 and a layer oftransparent, conventionally used PCR film 153 as shown in FIG. 2D. Thethickness of a sealing film may range from about 50 μm to about 255 μm.

As shown in FIG. 2A, the system may have one or more light sources 140.The light sources in a system may be the primary source of heat for thereaction vessel. The system may comprise one light source per system.Alternatively, the system may comprise more than one light source. Insome cases, the number of light sources in a system is the same as thenumber of intrusions in the transparent block. Each intrusion of thetransparent block may be in contact with one or more light sources. Insome cases, each intrusion of a transparent block may be in thermalcommunication with a single light source. In some cases, differentwells/intrusions may be able to maintain different thermal profilesbased on their light source. For instance, a well in a reaction vesselmay be maintained at 65° C. and another well may be maintained at 60° C.based on their light sources. The emitting area or chip size of thelight sources may cover the size of the intrusions in the transparentblock. Light sources may be light-emitting diode (LED), laser diode(LD), tungsten lamp, fluorescent lamp, halogen lamp, mercury lamp, xenonlamp, metal halide lamp or combinations thereof.

In some embodiments, the wavelength of the light sources may be in arange that does not coincide with the wavelength of fluorescent dyes.Non-limiting examples of wavelengths of light source include: 400 nm,405 nm, 440 nm, 445 nm, 460 nm, 650 nm, 720 nm, 850 nm and 950 nm.

In some cases, the heating rate of a light absorbing material may bedependent on the light source. Using a 3 W LED as an example of a lightsource, the heating rate of a light absorbing material by a light sourcemay be between 2° C./sec and 20° C./sec. In some cases, the heating ratemay be about 2° C./sec to about 20° C./sec. In some cases, the heatingrate may be at least about 5° C./sec. In some cases, the heating ratemay be at most about 20° C./sec. In some cases, the heating rate may beabout 2° C./sec to about 3° C./sec, about 5° C./sec to about 7° C./sec,about 5° C./sec to about 10° C./sec, about 5° C./sec to about 13°C./sec, about 5° C./sec to about 15° C./sec, about 5° C./sec to about18° C./sec, about 5° C./sec to about 20° C./sec, about 7° C./sec toabout 10° C./sec, about 7° C./sec to about 13° C./sec, about 7° C./secto about 15° C./sec, about 7° C./sec to about 18° C./sec, about 7°C./sec to about 20° C./sec, about 10° C./sec to about 13° C./sec, about10° C./sec to about 15° C./sec, about 10° C./sec to about 18° C./sec,about 10° C./sec to about 20 ° C./sec, about 13° C./sec to about 15°C./sec, about 13° C./sec to about 18° C./sec, about 13° C./sec to about20° C./sec, about 15° C./sec to about 18° C./sec, about 15° C./sec toabout 20° C./sec, or about 18° C./sec to about 20° C./sec. In somecases, the heating rate may be about 5° C./sec, about 7° C./sec, about10° C./sec, about 13° C./sec, about 15° C./sec, about 18° C./sec, orabout 20° C./sec. Heating rates may be different in higher or lowerpower light sources.

In some cases, the light-to-heat conversion efficiency may be dependenton the wavelength and light output power of a light source, thethickness of a light absorbing material and the distance between thelight source and the light absorbing material. In some cases, thelight-to-heat efficiency is at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90% or at least 95%.

A system may comprise an optical signal measurement apparatus (OSM) 160.OSM apparatus may comprise one or more excitation sources 161. In somecases, an apparatus may comprise one excitation source. In some cases,an apparatus may comprise more than one excitation source. Theexcitation source may be placed above the transparent block or on thesides of the transparent block. In some cases, excitation sources may beLEDs. The LEDs are excitation sources that may be used to generate lightat desired wavelengths to excite labels used for detecting nucleic acidproducts during real-time PCR, dissociation behavior during thermal meltanalysis, and/or nucleic acid related assays. The excitation sources maybe used to excite the one or more fluorescent dyes in the reactionvessel. Examples of fluorescent dyes that may be used may be anyfluorescent dye described herein and as known to one of ordinary skillin the art. Non-limiting examples of wavelengths of the excitationsources include 460 nm, 440 nm, 470 nm, 500 nm, 600 nm, 650 nm, 700 nm.

The optical measurement apparatus may further comprise one or moreoptical filters 162. The optical filters may be used to allow onlyselected wavelengths to reach a fluorescent dye in the reaction vesselsand wells in the apparatus. Optical filters could be used, at the outputof the LEDs/lasers or both to suppress any unwanted light (e.g. lightemitted by the LED aside from the center wavelength peak). The OSM maycomprise multiple filters such as a filter that may allow the excitationsource wavelength, a filter that allows the emission source wavelength,a filter to reduce or eliminate the light from the light source used forheating amongst others. Filters may include but are not limited to longpass filters for wavelengths between 400-560 nm, for example, 530 longpass filter, or short pass filters for wavelengths between 800-900 nm,for example a 840 nm short pass filter.

The OSM may further comprise one or more sensors or detectors 165 todetect the signal from the reaction vessel. The sensor may be a CMOSsensor, CCD sensor, photodiode or a spectrophotometer. The apparatus maycomprise one or more photodiode per well of the reaction vessel. Thesensor may be used to detect the modification of one or more nucleicacid targets.

The system may further comprise one or more embedded lenses 190 to focusthe light from the reaction vessel and direct it towards the sensor 165.FIG. 2E illustrates an example of an embedded lens 190 above a reactionvessel 130. Embedded lens 190 may be integrated with other elements ofthe optical system as described herein. There may be one sensor perreaction vessel. In some cases, there may be more than one lens perreaction vessel. Alternatively, there may be a lens for each well of thereaction vessel. The embedded lens may be placed between the lightsource and the transparent block. Alternatively, the embedded lens maybe placed between a detector region in the OSM and the reaction vessel.

The system may also comprise one or more temperature sensors (notshown). The one or more temperature sensors may be placed inside thereaction vessel, on the surface of the reaction vessel, embedded in thewall of the reaction vessel or on the surface of the light absorbinglayer. The system may comprise one temperature sensor per reactionvessel or the number of temperature sensors may be equal to the numberof wells in the reaction vessel. Temperature sensors may includethermocouples, IR temperature sensors, resistance temperature detectors,thermistor sensors and other sensors as are known to one of ordinaryskill in the art.

As shown in FIG. 2A, reagents 180 may be added to one or more wells of areaction vessel. Reagents may be lyophilized. Lyophilized reagents maybe coated with a hydrogel or paraffin wax. Samples and reagents may beadded to the wells in the reaction vessel using valves or ports (shownin FIG. 3). The reagents may be placed on to the wells individually orthrough a channel. The system may comprise of one or more channelnetworks. The one or more channel networks may be microfluidic channelnetworks. Sample or reagents may be added to individual wells or throughsuch a channel 330 as shown in FIG. 3. A cover plate or lid 157 can beutilized in conjunction with the microfluidic channel networksillustrated in FIG. 3.

The system in FIG. 3 and systems described in other embodiments maycomprise one or more valves. The wells of the reaction vessel may bepre-loaded with different reagent before the thermal cycling isinitiated. The one or more valves may prevent mixing of reagent solutionbetween the wells on the reaction vessel. Non-limiting examples ofvalves that may be used include solenoid valves, screw valves, pneumaticvalves, etc. In some cases, each well in the reaction vessel may haveaccess to one or more valves. Valves may be placed between each reactionwell, between the inlet and first wells and/or between the outlet andthe last wells in the reaction vessel.

Another embodiment of the system is illustrated in FIG. 4. Thetransparent block 110 may be placed above a base 115. The transparentblock may be coupled to side supports 116 and can be placed using theside supports. The side supports may assist in maintaining a certaindistance between the light source and the transparent block. System 100may comprise a transparent block 110 with a fluid circulation channel170. A cross section of the transparent block with the channel 170 isshown in FIG. 4. The fluid channel may be used to circulate any fluidsduring thermal cycling. Fluids may include, water, air, coolant liquidsand other fluids. The fluid flowing through the fluid circulationchannel may be transparent so as not to interfere with the lightemission from the light source 140. The fluid circulation channel may bedesigned to have a shape similar to the shape of the intrusions in thereaction vessel and the transparent block. During thermal cycling,different fluids may be cycled in the circulation channel. For example,in a cooling phase, water or a coolant liquid may be circulated to cooldown the reaction vessel. In some cases, air may be circulated duringthermal cycling. Fluids may be circulated using conventional pumps knownin the field.

Fluids may be circulated through the fluid circulation channel for atleast 1 second to at most 60 seconds. In some cases, fluid is circulatedin the channel for about 1 second to about 60 seconds. In some cases,fluid is circulated in the channel for at least about 1 second. In somecases, fluid is circulated in the channel for at most about 60 seconds.In some cases, fluid is circulated in the channel for about 1 second toabout 5 seconds, about 1 second to about 10 seconds, about 1 second toabout 20 seconds, about 1 second to about 30 seconds, about 1 second toabout 40 seconds, about 1 second to about 50 seconds, about 1 second toabout 60 seconds, about 5 seconds to about 10 seconds, about 5 secondsto about 20 seconds, about 5 seconds to about 30 seconds, about 5seconds to about 40 seconds, about 5 seconds to about 50 seconds, about5 seconds to about 60 seconds, about 10 seconds to about 20 seconds,about 10 seconds to about 30 seconds, about 10 seconds to about 40seconds, about 10 seconds to about 50 seconds, about 10 seconds to about60 seconds, about 20 seconds to about 30 seconds, about 20 seconds toabout 40 seconds, about 20 seconds to about 50 seconds, about 20 secondsto about 60 seconds, about 30 seconds to about 40 seconds, about 30seconds to about 50 seconds, about 30 seconds to about 60 seconds, about40 seconds to about 50 seconds, about 40 seconds to about 60 seconds, orabout 50 seconds to about 60 seconds. In some cases, fluid is circulatedin the channel for about 1 second, about 5 seconds, about 10 seconds,about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds,or about 60 seconds.

In this example, the light absorbing material 120 is placed on theintrusions of the transparent block 110 instead of inside the reactionvessel 130. In such cases, any conventional PCR plate may be used as areaction vessel combined with a conventional sealing film or a sealingfilm described herein. In other examples, the light absorbing layer 120may be placed in the reaction vessel. As discussed in relation to FIG.2C, a layer of light absorbing material 152 may be incorporated as anelement of a sealing film, illustrated in FIG. 4 as sealing film 150.

Another embodiment of the system is shown in FIG. 5. Transparent block110 with a light absorbing layer placed in the intrusions may have areaction vessel sealed with a sealing film 150. In addition, thetransparent block may be covered on the outside with a high refractiveindex film 111. When light from the light source or the excitationsource enters the transparent block covered with a film with highrefractive index, the angle of refraction may be smaller than the angleof incidence and the light may be refracted towards the normal of thesurface for a more uniform transmission of light in to the wells of thereaction vessel.

Also illustrated in FIG. 5 is an embodiment of the OSM apparatus 160.The OSM apparatus may comprise one or more excitation sources 161. Theone or more excitation sources may be configured to excite one or morefluorescent dyes used in the nucleic acid modification assays.Fluorescent dyes and their corresponding excitation sources may be anycommonly used ones as are known to one of ordinary skill in the art.More than one type of excitation sources may be used in one OSMapparatus. For instance, the OSM apparatus may comprise an excitationsource with the wavelength 460 nm to excite dyes such as FAM, SYBRgreen, etc. in addition to an excitation source with a wavelength of 440nm to excite dyes such as LC Green.

The OSM apparatus may also comprise one or more optical filters. In thisexample, an optical filter 162 may be placed in front of the excitationsource. This optical source may be configured to only allow theexcitation light wavelength. For example, for an excitation source ofwavelength 460 nm, an optical filter 162 may be a 480 nm short passfilter. Other optical filters known in the art may also be used. Inaddition to the optical filter 162, the OSM apparatus may also comprisea first filter 164 and a second filter 163. The second filter 163 placedabove the reaction vessel may be used as an elimination filter to removethe light from light source 140. For instance, the second filter 163 maybe a filter specific for elimination of 800-850 nm wavelength. Thesecond filter may be a Distributed Bragg reflector (DBR) with highreflectivity over the emission wavelength of the light source. The firstfilter may be used as an emission filter. The emission filter may onlyallow light emitted from the reaction vessel as a result of theexcitation of a fluorescent dye in the reaction vessel. For instance,the first filter or the emission filter may be a long pass filterspecific to allow light with wavelength of 470-530 nm. Any conventionalfilters, known to a person of ordinary skill in the art may be used. TheOSM apparatus may also comprise sensors 165 as described previouslyherein.

Another embodiment of the system is illustrated in FIGS. 6-8. FIG. 6 isan exploded view of the system in a cross-section view. The transparentblock 110 may comprise intrusions (not shown here) and a fluidcirculation channel 170 as previously described. Components of thesystem may be coupled to supports that are not shown in thecross-section illustrated in FIG. 6. A reaction vessel 130, as describedpreviously may be removably placed on the transparent block 110. In someembodiments, the light absorbing material 120 may be removably placed onthe reaction vessel 130. The sample may be placed in the reaction vesselin direct contact with the light absorbing layer. A sealing plate 610may be removably placed on the reaction vessel 130.

The sealing plate 610 may be made of the same material as the reactionvessel. The sealing plate may have intrusions 612 shaped similar to thewells of the reaction vessel as shown in FIG. 6. Portions or all of thebottom surface of the sealing plate may be covered with a lightabsorbing material 121. The sealing plate may also comprise spacers 155.

FIG. 7 shows the mechanism of using a sealing plate 610 with the system.A transparent block with reaction wells 130 with the wells covered witha light absorbing layer 120 may be placed on the transparent block.Reagents and samples may be placed in the wells of the reaction well130. A sealing plate 610 with intrusions, the shape of the wells of thereaction vessel, may be placed on top of the reaction vessels. Thebottom layer of the sealing plate 610 may comprise a layer of lightabsorbing material 121. The corners of the sealing plate may comprise anadhesive layer 151 as illustrated in FIG. 8 to seal the reaction vesseland the sealing plate. The sealing plate may also comprise spacers 155as illustrated in FIG. 8. The spacers can be used to maintain a spacebetween the sealing plate and the reaction vessel.

Upon sealing, sample and reagents 180 may get uniformly distributed inthe reaction vessel covered on both sides with light absorbing layers120 and 121. A reaction vessel may comprise a lower light absorbinglayer and an upper light absorbing layer wherein the reaction vessel maybe defined to be between the two light absorbing layers. The reactionvessel and the sealing plate may be used as supports for the lightabsorbing layers with a first light absorbing material disposed on afirst support to define a reaction well and a second light absorbingmaterial disposed on a second support opposite the first support as partof the sealing plate.

The volume of the sample and reagents used in such a system may beconfigured to leave air gaps between the sample and the corners of thesealing plate. The system may have a fluid circulation channel (notshown here) for uniform cooling of the reaction vessel.

FIG. 8 illustrates the embodiments of FIG. 7 in greater depth. Thebottom plate may comprise reaction wells (such as 131) covered with alayer of light absorbing material 120. The thickness of the lightabsorbing layer 120 may be from 1 nm to 1 mm. In this example, thethickness of the reaction well (131) is 250 μm. The wells in thereaction well may have a diameter ranging from 1 mm to 10 mm. In thisexample, the diameter of the reaction well shown is 4 mm. The reactionwell may be used to contain a sample volume from 1 μl to 20 μl.

Sealing plate 610 may comprise one or more intrusions 612 as illustratedin FIG. 6. The diameter of the intrusion in the sealing plate may besmaller than the diameter of the wells in the reaction well to leavespace for the sample and reagents. The diameter of the intrusions in thesealing plate may be from 0.5 mm to 8 mm. In this example, the diameterof the sealing plate is 3 mm. Sealing plate 150 comprising a layer oflight absorbing material 121 may be placed on top of the reaction well.The thickness of the light absorbing layer may be the same as layer 120.In some embodiments, the thickness of the light absorbing layer may beless or more than the thickness of layer 120. In some cases, layers 120and 121 may be made of the same material. For instance, in one exampleboth layer 120 and 121 are made of a metal such as gold or chromium. Insome alternative embodiments, layers 120 and 121 may be made ofdifferent materials. For instance, layer 120 may be made of a metal suchas gold and layer 121 may be made of a carbon based material.

Modifications of nucleic acids may comprise identifying a target nucleicacid from a sample. A PCR reaction using the systems discussed hereinmay be performed for such modifications. A sample containing nucleicacids may be added to reagents (lyophilized, stabilized or in asolution) in the presence of a buffer solution. The mixture may thenundergo thermal cycling through a range of temperatures to complete theamplification process. Thermal cycling may include multiple cycles suchas a denaturation cycle, an annealing cycle, an elongation cycle and/oran incubation cycle.

In some embodiments, the reagents 180 may be placed in the wells of thereaction well 130 of the systems discussed herein. The reagents may bein the form of lyophilized beads or pellets. Stabilization of thereagents may be performed using a hydrogel or paraffin wax. The hydrogelor paraffin may have a melting temperature higher than room temperature.Reagents and samples may be loaded on to the wells of the reaction wellusing channel, pumps and valves as described herein or as are known toone of ordinary skill in the art.

Upon loading and sealing, the system may generate an amplified productthrough thermal cycling. Thermal cycling may comprise one or more cyclesof incubating a reaction mixture at a denaturation temperature for adenaturation time period followed by incubating the mixture at anannealing temperature for an annealing time period further followed byincubating the mixture at an elongation temperature for an elongationtime period. A system may heat the wells of the reaction well 130 (notshown) by using one or more light sources 140 (not shown) as previouslydescribed. Focused light by lens between the one or more light sourcesand the reaction well may be used also. The embedded lens may be used tofocus emission from the fluorescent dye integrated in the reactionvessel/wells. For the cooling of the sample and reagents, the one ormore light sources may be turned off for a cooling time period. In somecases, a fluid circulation channel 170 may be used as previouslydescribed for the cooling of the reagents and samples in the wells ofthe reaction well.

Amplification a sample may be performed by using the systems describedpreviously to perform one or more thermal cycles comprising adenaturation cycle, an annealing cycle and an elongation cycle. The timein which an amplification reaction may yield a detectable result in theform of an amplified product may vary depending on the target nucleicacid, the sample, the reagents used and the protocol for PCR. In somecases, an amplification process may be performed in less than 1 minute.In some cases, an amplification process may be performed in about 1minute to about 40 minutes. In some cases, an amplification process maybe performed in at least about 1 minute. In some cases, an amplificationprocess may be performed in at most about 40 minutes. In some cases, anamplification process may be performed in about 1 minute to about 5minutes, about 1 minute to about 10 minutes, about 1 minute to about 15minutes, about 1 minute to about 20 minutes, about 1 minute to about 25minutes, about 1 minute to about 30 minutes, about 1 minute to about 35minutes, about 1 minute to about 40 minutes, about 5 minutes to about 10minutes, about 5 minutes to about 15 minutes, about 5 minutes to about20 minutes, about 5 minutes to about 25 minutes, about 5 minutes toabout 30 minutes, about 5 minutes to about 35 minutes, about 5 minutesto about 40 minutes, about 10 minutes to about 15 minutes, about 10minutes to about 20 minutes, about 10 minutes to about 25 minutes, about10 minutes to about 30 minutes, about 10 minutes to about 35 minutes,about 10 minutes to about 40 minutes, about 15 minutes to about 20minutes, about 15 minutes to about 25 minutes, about 15 minutes to about30 minutes, about 15 minutes to about 35 minutes, about 15 minutes toabout 40 minutes, about 20 minutes to about 25 minutes, about 20 minutesto about 30 minutes, about 20 minutes to about 35 minutes, about 20minutes to about 40 minutes, about 25 minutes to about 30 minutes, about25 minutes to about 35 minutes, about 25 minutes to about 40 minutes,about 30 minutes to about 35 minutes, about 30 minutes to about 40minutes, or about 35 minutes to about 40 minutes. In some cases, anamplification process may be performed in about 1 minute, about 5minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25minutes, about 30 minutes, about 35 minutes, or about 40 minutes.

In some cases, amplification of a sample may be performed by repeatingthe thermal cycle 5 to 40 times. In some cases, the thermal cycle may berepeated at least 5 times. In some cases, the thermal cycle may berepeated at most 60 times. In some cases, the thermal cycle may berepeated 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35times 40 times, 45 times, 50 times, 55 times or 60 times.

A thermal cycle may be completed in a thermal cycling time period. Insome cases, a thermal cycling time period may range from 2 seconds to 60seconds per cycle. In some cases, a thermal cycle may be completed inabout 2 seconds to about 60 seconds. In some cases, a thermal cycle maybe completed in at least about 2 seconds. In some cases, a thermal cyclemay be completed in at most about 60 seconds. In some cases, a thermalcycle may be completed in about 2 seconds to about 5 seconds, about 2seconds to about 10 seconds, about 2 seconds to about 20 seconds, about2 seconds to about 40 seconds, about 2 seconds to about 60 seconds,about 5 seconds to about 10 seconds, about 5 seconds to about 20seconds, about 5 seconds to about 40 seconds, about 5 seconds to about60 seconds, about 10 seconds to about 20 seconds, about 10 seconds toabout 40 seconds, about 10 seconds to about 60 seconds, about 20 secondsto about 40 seconds, about 20 seconds to about 60 seconds, or about 40seconds to about 60 seconds. In some cases, a thermal cycle may becompleted in about 2 seconds, about 5 seconds, about 10 seconds, about20 seconds, about 40 seconds, or about 60 seconds.

The temperature and time period of the denaturation cycle may bedependent on the properties sample to be identified, the reagents andthe amplification protocol being used. A denaturation cycle may beperformed at temperatures ranging from about 80° C. to about 110° C. Adenaturation cycle may be performed at a temperature of at least about80° C. A denaturation cycle may be performed at a temperature of at mostabout 110° C. A denaturation cycle may be performed at a temperature ofabout 80° C. to about 85° C., about 80° C. to about 90° C., about 80° C.to about 95° C., about 80° C. to about 100° C., about 80° C. to about105° C., about 80° C. to about 110° C., about 85° C. to about 90° C.,about 85° C. to about 95° C., about 85° C. to about 100° C., about 85°C. to about 105° C., about 85° C. to about 110° C., about 90° C. toabout 95° C., about 90° C. to about 100° C., about 90° C. to about 105°C., about 90° C. to about 110° C., about 95° C. to about 100° C., about95° C. to about 105° C., about 95° C. to about 110° C., about 100° C. toabout 105° C., about 100° C. to about 110° C., or about 105° C. to about110° C. A denaturation cycle may be performed at a temperature of about80° C., about 85° C., about 90° C., about 95° C., about 100° C., about105° C., or about 110° C.

In some cases, the time period of a denaturation cycle may be less thanabout 1 second. In some cases, the time period of a denaturation cyclemay be at most about 100 seconds. In some cases, the time period of adenaturation cycle may be about 0 second to 1 second, about 1 second toabout 5 seconds, about 1 second to about 10 seconds, about 1 second toabout 20 seconds, about 1 second to about 40 seconds, about 1 second toabout 60 seconds, about 1 second to about 100 seconds, about 5 secondsto about 10 seconds, about 5 seconds to about 20 seconds, about 5seconds to about 40 seconds, about 5 seconds to about 60 seconds, about5 seconds to about 100 seconds, about 10 seconds to about 20 seconds,about 10 seconds to about 40 seconds, about 10 seconds to about 60seconds, about 10 seconds to about 100 seconds, about 20 seconds toabout 40 seconds, about 20 seconds to about 60 seconds, about 20 secondsto about 100 seconds, about 40 seconds to about 60 seconds, about 40seconds to about 100 seconds, or about 60 seconds to about 100 seconds.In some cases, the time period of a denaturation cycle may be less thanabout 1 second, about 5 seconds, about 10 seconds, about 20 seconds,about 40 seconds, about 60 seconds, or about 100 seconds.

The temperature and time period of the annealing and elongation cyclesmay be dependent on the properties sample to be identified, the reagentsand the amplification protocol being used. An annealing and/orelongation cycle may be performed at a temperature of about 40° C. toabout 70° C. An annealing and/or elongation cycle may be performed at atemperature of at least about 40° C. An annealing and/or elongationcycle may be performed at a temperature of at most about 70° C. Anannealing and/or elongation cycle may be performed at a temperature ofabout 40° C. to about 45° C., about 40° C. to about 50° C., about 40° C.to about 55° C., about 40° C. to about 60° C., about 40° C. to about 65°C., about 40° C. to about 70° C., about 45° C. to about 50° C., about45° C. to about 55° C., about 45° C. to about 60° C., about 45° C. toabout 65° C., about 45° C. to about 70° C., about 50° C. to about 55°C., about 50° C. to about 60° C., about 50° C. to about 65° C., about50° C. to about 70° C., about 55° C. to about 60° C., about 55° C. toabout 65° C., about 55° C. to about 70° C., about 60° C. to about 65°C., about 60° C. to about 70° C., or about 65° C. to about 70° C. Anannealing and/or elongation cycle may be performed at a temperature ofabout 40° C., about 45° C., about 50° C., about 55° C., about 60° C.,about 65° C., or about 70° C.

In some cases, the time period of an annealing and/or elongation cyclemay be less than about 1 second. In some cases, the time period of anannealing and/or elongation cycle may be at most about 60 seconds. Insome cases, the time period of an annealing and/or elongation cycle maybe about 0 seconds to 1 seconds, about 1 second to about 5 seconds,about 1 second to about 10 seconds, about 1 second to about 20 seconds,about 1 second to about 40 seconds, about 1 second to about 60 seconds,about 5 seconds to about 10 seconds, about 5 seconds to about 20seconds, about 5 seconds to about 40 seconds, about 5 seconds to about60 seconds, about 10 seconds to about 20 seconds, about 10 seconds toabout 40 seconds, about 10 seconds to about 60 seconds, about 20 secondsto about 40 seconds, about 20 seconds to about 60 seconds, or about 40seconds to about 60 seconds. In some cases, the time period of anannealing and/or elongation cycle may be less than about 1 second, about5 seconds, about 10 seconds, about 20 seconds, about 40 seconds, orabout 60 seconds.

In some cases, a cooling cycle may be performed between the denaturationcycle and annealing and/or elongation cycles. In some cases, a coolingcycle may be performed for about 1 second to about 60 seconds. In somecases, a cooling cycle may be performed for at least about 1 second. Insome cases, a cooling cycle may be performed for at most about 60seconds. In some cases, a cooling cycle may be performed for about 1second to about 5 seconds, about 1 second to about 10 seconds, about 1second to about 20 seconds, about 1 second to about 30 seconds, about 1second to about 40 seconds, about 1 second to about 50 seconds, about 1second to about 60 seconds, about 5 seconds to about 10 seconds, about 5seconds to about 20 seconds, about 5 seconds to about 30 seconds, about5 seconds to about 40 seconds, about 5 seconds to about 50 seconds,about 5 seconds to about 60 seconds, about 10 seconds to about 20seconds, about 10 seconds to about 30 seconds, about 10 seconds to about40 seconds, about 10 seconds to about 50 seconds, about 10 seconds toabout 60 seconds, about 20 seconds to about 30 seconds, about 20 secondsto about 40 seconds, about 20 seconds to about 50 seconds, about 20seconds to about 60 seconds, about 30 seconds to about 40 seconds, about30 seconds to about 50 seconds, about 30 seconds to about 60 seconds,about 40 seconds to about 50 seconds, about 40 seconds to about 60seconds, or about 50 seconds to about 60 seconds. In some cases, acooling cycle may be performed for about 1 second, about 5 seconds,about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds,about 50 seconds, or about 60 seconds.

Detection of the amplified product using OSM 160 as described previouslymay be performed at various stages of the amplification process. In somecases, the detection of an amplified product may be performed at the endof the amplification process. In some cases, the detection of theamplified product may be performed during a thermal cycle.Alternatively, in some cases, detection may be performed at the end ofeach thermal cycle. In addition to the detection methods describedherein, detection of an amplified product may be performed using gelelectrophoresis, capillary electrophoresis, sequencing, short tandemrepeat analysis and other methods as are known to one of ordinary skillin the art.

Light absorbing material placed in the system for modification ofnucleic acids, as described in any of the embodiments herein, may be inthe form of a solid shape as shown in FIG. 9A. FIG. 9A shows the lightabsorbing material as a circle but it may be in different shapes, suchas the shape of the reaction wells or the intrusions in the transparentblock. In some cases, the light absorbing material may have one or moreopen areas. In some cases, the light absorbing material may have oneopen area 910 in the center, such as shown in FIG. 9B. The open area 910in the center of the light absorbing material, which can representeither light absorbing material 120 or light absorbing material 121 asillustrated in FIG. 2A, or both, may be used to direct light from anexcitation source for the detection of fluorescence in the reactionmixture. In some cases, the light absorbing material may have more thanone open areas as shown in FIG. 9C. Multiple open areas 912 may be usedto direct different excitation sources to the fluorescent material inthe reaction mixture or the same excitation source light may be directedusing multiple open areas in the light absorbing material. The openareas in a light absorbing material may only be on the light absorbingmaterials in the reaction vessel. In some cases, light absorbingmaterial in the sealing film or sealing plate may also have one or moreopen areas. In some cases, light absorbing material in both a reactionwell and in a sealing film/plate may have open areas.

In some cases, the percentage of open area in a light absorbing materialmay be about 1% to about 90%. In some cases, the percentage of open areain a light absorbing material may be at least about 1%. In some cases,the percentage of open area in a light absorbing material may be at mostabout 90%. In some cases, the percentage of open area in a lightabsorbing material may be about 1% to about 10%, about 1% to about 20%,about 1% to about 50%, about 1% to about 70%, about 1% to about 90%,about 10% to about 20%, about 10% to about 50%, about 10% to about 70%,about 10% to about 90%, about 20% to about 50%, about 20% to about 70%,about 20% to about 90%, about 50% to about 70%, about 50% to about 90%,or about 70% to about 90%. In some cases, the percentage of open area ina light absorbing material may be about 1%, about 10%, about 20%, about50%, about 70%, or about 90%.

Operation of the light source may be performed in a continuous or apulsed manner. In some cases, the operation of the light source iscontinuous, for instance as shown in FIG. 10. In some cases, for everyheating cycle including denaturation, annealing and extension cycles,the light source can be operated to increase temperature fordenaturation. This may be performed by applying a certain injectioncurrent to the light source. To maintain the temperature forannealing/extension, the injection current of the light source may beadjusted without turning off the light source. The light source may beturned off during the cooling cycle.

In some cases, the light source may be operated in a pulsed manner, asshown in FIG. 10. For every heating cycle, the light source may beturned on and off repeatedly at short intervals. In some cases, theintervals for each cycle can be modified for instance, the denaturationcycle may have shorter intervals of pulses as compared to a lowerannealing temperature. In some cases, the injection current to the lightsource may be modified during a pulsing cycle. The light source may beturned off during the cooling cycle.

In some embodiments, operation of the excitation source may be pulsed.In some cases, the pulsed operation of the excitation source may beperformed in an alternating manner as compared to the pulsing of thelight source as shown in FIG. 11A. As the light source is pulsed to anoff cycle, the excitation source may be turned on. As the light sourceis pulsed to an on cycle, the excitation source may be turned off. Insome embodiments, detection of a signal may be performed parallel to thepulsing of the excitation source. The sensors detecting a fluorescentsignal from the reaction wells may collect data in a pulsing mannerparallel to the excitation source as shown in FIG. 11A. This may beperformed to reduce or avoid optical interference between the lightsource and the excitation source or the interference between the lightsource and the fluorescence in the reaction wells which may be theresult of a nucleic acid modification.

In other embodiments, operation of the excitation source may becontinuous as illustrated in FIG. 11B and discussed in relation to FIG.10. Thus, in some cases, the pulsed operation of the light source may beperformed in an alternating manner while the operation of the excitationsource is continuous. In some embodiments, detection of a signal may beperformed parallel to the pulsing of the excitation source. The sensorsdetecting a fluorescent signal from the reaction wells may collect datain a pulsing manner parallel to the light source as shown in FIG. 11B.This may be performed to reduce or avoid optical interference betweenthe light source and the excitation source or the interference betweenthe light source and the fluorescence in the reaction wells which may bethe result of a nucleic acid modification.

Sample Preparation

The systems for nucleic acid modification may in some cases include asystem or module for sample preparation. The sample preparation systemmay be used for the concentration of a cell of interest. The cells ofinterest may be any specific target cell. For instance, the cell ofinterest may be red blood cells, platelets, leukocytes, infectious cellssuch as pathogens amongst other cell types. The sample preparation celltype may also be used to extract and purify nucleic acids from targetcells. The sample used for the concentration of the target cell type maybe any sample type described herein.

In some cases, the sample preparation system may be able to concentratea cell type of interest from a biological sample, extract and purifynucleic acids from a sample in less than 15 minutes. In some cases, thesample preparation system may be able to extract and purify nucleicacids from a biological sample in less than 15 minutes, less than 12minutes, less than 10 minutes, less than 8 minutes, less than 5 minutes,less than 2 minutes or less than 1 minute.

Referring to FIG. 12, a system for sample preparation is shown. Samplepreparation system 1200 may have multiple fluid compartments, such as asample compartment 1210 to collect a biological sample, one or more washbuffer compartments 1220 and 1230, a waste compartment 1240 and anelution buffer compartment 1250. The one or more compartments of thesample preparation system may be connected to a collection area 1261with a cover 1260 through various microfluidic channels, reservoirs andthrough-holes. The microfluidic system may be in the form of areplaceable cartridge in the sample preparation system.

A sample compartment may comprise a sample inlet 1215. The sample inlet1215 may comprise a pre-filter for filtering out cells and debris andcrystals by size. The pre-filter may be above the through-hole 1211instead of at the sample inlet 1215. In some cases the pre-filter mayhave a pore size of 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm or 10 μm.The pre-filter may be able to remove non-target materials such assediments and larger cells from a biological sample such as a urinesample.

Each compartment may have an air inlet. Each air inlet of a liquidcontainer may be serially connected to a micro-solenoid valve and thento a pump. As shown in FIG. 12, each fluid compartment may have theirspecific air-inlets. For the sample compartment, sample inlet 1215 mayalso act as the air inlet. In some cases, the sample compartment mayhave a separate air-inlet. Also shown in FIG. 12 are air-inlets for washbuffer compartments 1224 and 1234, air-inlet 1243 for the wastecompartment and air-inlet 1234 for the elution buffer compartment. Theliquid outlets may be directly connected to the designated microchannelsconnected to each compartment. Microfluidic channels may be made of PDMSPMMA, COC, other conventional polymers and/or SU-8 silicon wafer.

The microfluidic channels may all be in a single layer or they may bedivided over multiple layers of channels and collection areas,reservoirs or through-holes. For instance, in FIG. 12, shown are twolayers of microfluidic channels, a first layer and a second layer.Multiple compartments in the sample preparation system may span overmultiple layers of the microfluidic system. As shown in FIG. 12, asample compartment may be connected to a through-hole 1211 in the firstlayer and through-hole 1211 may be connected to the reservoir 1212 inthe second layer with a microfluidic channel 1213 which may be used toconnect the sample compartment to the collection area 1261 with a cover1260. Through-holes and reservoirs in the microfluidic system may be ofthe same shape and size. Alternatively, the reservoirs may in somecases, be larger than the through-holes. Through-holes and theircorresponding reservoirs may be aligned together.

Similarly other compartments may also be connected. In FIG. 12, washcompartments 1220 and 1230 may be connected to through-holes 1221 or1231 respectively, in the first layer. Through-holes 1221 and 1231 maybe connected to reservoirs 1222 or 1232 in the second layer and connectto the collection area 1261 through channels 1223 and 1233 respectively.The waste compartment may also be connected to the microfluidic systemusing through-holes and channels. As shown in FIG. 12, waste compartment1240 may be connected to through-hole 1241 and also connects to thecollection area 1261 through a channel 1242. Also shown in FIG. 12 isthe elution buffer compartment 1250 connected to the microfluidic systemby through-hole 1251 in the first layer, reservoir 1252 in the secondlayer and channel 1253 to connect the elution buffer compartment to thecollection area 1261. FIG. 12 represents a system with one collectionarea and one channel leading from each compartment to the collectionarea but the sample preparation system may comprise multiple collectionareas connected to multiple channels for each compartment.

Collection area 1261 may be in the shape of a well as shown in FIG. 12and covered with a cover 1260. Collection area 1261 may comprise afilter 1270 between the cover 1260 and collection area 1261 for theentrapment and processing of one or more target cells. The samplepreparation system may comprise more than one filter. A target cell typemay be entrapped and treated for nucleic acid extraction andpurification on one or more filters.

Collection area 1261 may also be covered with a light absorbing material1280 for the photothermal lysis of target cells. In some cases, thecollection area 1261 is covered with a light absorbing material 1280 anda filter 1270. The filter 1270 may be placed on top of the lightabsorbing material 1280. Alternatively, the light absorbing material1280 may be below the collection area 1261. In such cases, the filter1270 may be on the collection area. Photothermal lysis of a target cellmay be performed by using a light source for the conversion of light toheat. Light sources may be any light sources described elsewhere hereinand may be placed below the microfluidic system. In some cases, a filter1270 entrapping one or more target cells may be placed above or near alight source and a light absorbing material 1280. Light absorbingmaterial may be any light absorbing material described elsewhere herein.In FIG. 12, the microfluidic network is shown to be connected directlyto a PCR compartment 1290 by channel 1291. In other examples, themicrofluidic network may not be in the same cartridge as shown in FIG.12. In such example, different air-inlets and pumps may be used for themovement of the sample nucleic acids to reaction wells as describedelsewhere herein.

The sample preparation system may have one or more air inlets. Thesample preparation system may have one or more pneumatic control valves.The pneumatic control valves may be a part of a pneumatic control systemfor sample fluid actuation. Pressurized air and control valves may beused for the movement of fluids from one reservoir to the other. Aschematic of a sample processing using valves and air-inlets is shown inFIG. 13.

Referring to FIGS. 12 and 13A, after adding sample solution to thesample compartment, solenoid valves connected to the air-inlet andreservoir of the sample (1211, 1212) and waste (1241) compartments maybe opened and pressurized air may be allowed in through air-inlet 1215and through-hole 1211 of the sample compartment so that the pressure isonly applied to the sample compartment. All of the other air-inlets maybe closed so that the sample solution only passes through microfluidicchannels 1213 and 1242. These channels may consist of two microfluidiclayers, top and bottom layers. The channel 1213 connected to the samplecompartment may be located in the second microfluidic layer (2nd layershown in FIG. 12) and the channel 1242 on the waste side is located inthe top layer (1st layer shown in FIG. 12). Overlapping between thesechannels may be the collection area 1261 comprising a filter 1270 toselectively isolate target cells. Filter 1270 may have a pore size thatis specific for the target cells. Afterwards, the sample and wastecompartments and their corresponding air-inlets may be closed by thesolenoid valves for the next step. The sample compartment may have oneor more electrodes placed around the compartment to change the pH of thesolution by applying different voltages.

Referring to FIG. 13B, a wash step is shown. Wash buffer A compartment1220 and waste compartment 1240 may be opened by solenoid valvesconnected to air-inlets or reservoir of the wash buffer A (1224, 1222)and waste (1243) compartments. All of the other air-inlets may be closedby the corresponding valves so that the wash buffer A solution onlypasses through microfluidic channels 1223 and 1242. Pressurized air maybe applied to the wash buffer A compartment through air-inlet 1224 toinitiate the flow. These channels may be located in the samemicrofluidic layers shown in FIG. 13A. The reservoir 1222 connected tothe wash buffer A compartment in FIG. 13B may be located in the firstlayer and the channel 1242 on the waste side may be located in thesecond layer. Overlapping between these channels may be the collectionarea 1261 and filter 1270 as shown in FIGS. 12 and 13A to wash outunwanted materials trapped in the filter during the target cellisolation step. Afterwards, the wash buffer A (1220) and waste (1240)compartments and their corresponding air-inlets may be closed by thesolenoid valves for the next step.

Referring to FIGS. 12 and 13C, a second wash step is shown. Wash bufferB compartment 1230 and waste compartment 1240 may be opened by solenoidvalves connected to air-inlets of the wash buffer B (1234) and waste(1243) compartments. All of the other air-inlets may be closed by thecorresponding valves so that the wash buffer B solution only passesthrough microfluidic channels 1233 and 1242. Pressurized air may beapplied to the wash buffer B compartment through air-inlet 1234 toinitiate the flow. These microfluidic channels may be located in thesame layers as shown earlier. The channel 1233 connected to the washbuffer B compartment 1230 may be located in the first layer and thechannel 1242 on the waste side may be located in the second layer.Overlapping between these channels may be the collection area 1261 andthe same filter 1270 to wash out unnecessary materials in the filterunit. Afterwards, the wash buffer B and waste compartments may be closedby the solenoid valves for the next step. The waste compartment maycomprise an absorbing porous paper, fabric or sponges to prevent re-fluxof fluid.

Referring to FIG. 13D, a next step as photothermal lysis of target cellsis shown. During this process, all of the valves may be closed. Thetrapped target cells in the filter may be thermally lysed using a lightsource to heat the light-absorbing film located on the collection areaor the light absorbing material located below the collection area. Thecollection area may be thermally connected to one or more light sources.

Referring to FIGS. 12 and 13E, an elution of nucleic acids step isshown. The lysed sample may be transferred to the photonic PCRcompartment. The elution step may be initiated by opening thethrough-holes 1251 and reservoir 1252 of the elution buffer compartment1250 and applying pressurized air to the same compartment usingair-inlet 1254. All of the other air-inlets may be closed so that theeluted sample only passes through channels 1253 (elution buffer channel)and 1291 (PCR compartment). The channel 1253 connected to the elutionbuffer compartment 1250 may be located in the first layer and thechannel 1291 on the PCR compartment 1290 may be located in the secondlayer. Overlapping between these channels may be the same filter 1270 inthe filter unit. Afterwards, all of the air-inlets may be closed by thesolenoid valves for the PCR process. The elution compartment may haveone or more electrodes placed around the compartment to change the pH ofthe solution by applying different voltages. In FIGS. 13A-E, the PCRunit 1290 has been shown to have four reaction wells but in some cases,the PCR unit may have more wells as described elsewhere herein as shownin FIG. 14. The sample preparation module with PCR reaction wellsillustrated in FIG. 14 shares common elements with the samplepreparation module with PCR reaction wells illustrated in FIG. 12 andthe description provided in relation to FIG. 12 is applicable to FIG. 14as appropriate. In FIG. 14, the number of PCR reaction wells is greaterthan the four PCR wells illustrated in FIGS. 12 and 13E.

The systems and methods described herein may be used to detect targetcells in a biological sample. The detection limit for an assay using themethods and systems described herein may be as low as 2 copies of DNA.The detection limit may be 2 copies of DNA, 5 copies of DNA, 10 copiesof DNA or 20 copies of DNA in a biological sample.

In some cases, the systems and methods herein may be used to detecttarget cells in a biological sample. The detection limit for an assayusing the methods and systems described herein may be as low as 2 CFU/mlin a biological sample. In some cases, the detection rate is as low as 2CFU/ml, 5, 7 CFU/ml, 10 CFU/ml, 12 CFU/ml, 15 CFU/ml, 20 CFU/ml or 25CFU/ml in a biological sample.

Digital Processing Device

In some embodiments, the platforms, systems, media, and methodsdescribed herein include a digital processing device, or use of thesame. In further embodiments, the digital processing device includes oneor more hardware central processing units (CPUs) or general purposegraphics processing units (GPGPUs) that carry out the device'sfunctions. In still further embodiments, the digital processing devicefurther comprises an operating system configured to perform executableinstructions. In some embodiments, the digital processing device isoptionally connected to a computer network. In further embodiments, thedigital processing device is optionally connected to the Internet suchthat it accesses the World Wide Web. In still further embodiments, thedigital processing device is optionally connected to a cloud computinginfrastructure. In other embodiments, the digital processing device isoptionally connected to an intranet. In other embodiments, the digitalprocessing device is optionally connected to a data storage device.

In accordance with the description herein, suitable digital processingdevices include, by way of non-limiting examples, server computers,desktop computers, laptop computers, notebook computers, sub-notebookcomputers, netbook computers, netpad computers, set-top computers, mediastreaming devices, handheld computers, Internet appliances, mobilesmartphones, tablet computers, personal digital assistants, video gameconsoles, and vehicles. Those of skill in the art will recognize thatmany smartphones are suitable for use in the system described herein.Those of skill in the art will also recognize that select televisions,video players, and digital music players with optional computer networkconnectivity are suitable for use in the system described herein.Suitable tablet computers include those with booklet, slate, andconvertible configurations, known to those of skill in the art.

In some embodiments, the digital processing device includes an operatingsystem configured to perform executable instructions. The operatingsystem is, for example, software, including programs and data, whichmanages the device's hardware and provides services for execution ofapplications. Those of skill in the art will recognize that suitableserver operating systems include, by way of non-limiting examples,FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle®Solaris®, Windows Server®, and Novell® NetWare®. Those of skill in theart will recognize that suitable personal computer operating systemsinclude, by way of non-limiting examples, Microsoft® Windows®, Apple®Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU/Linux®. Insome embodiments, the operating system is provided by cloud computing.Those of skill in the art will also recognize that suitable mobile smartphone operating systems include, by way of non-limiting examples, Nokia®Symbian® OS, Apple® iOS®, Research In Motion® BlackBerry OS®, Google®Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS,Linux®, and Palm® WebOS®. Those of skill in the art will also recognizethat suitable media streaming device operating systems include, by wayof non-limiting examples, Apple TV®, Roku®, Boxee®, Google TV®, GoogleChromecast®, Amazon Fire®, and Samsung® HomeSync®. Those of skill in theart will also recognize that suitable video game console operatingsystems include, by way of non-limiting examples, Sony® PS3®, Sony®PS4®, Microsoft® Xbox 360®, Microsoft Xbox One, Nintendo® Wii®,Nintendo® Wii U®, and Ouya®.

In some embodiments, the device includes a storage and/or memory device.The storage and/or memory device is one or more physical apparatusesused to store data or programs on a temporary or permanent basis. Insome embodiments, the device is volatile memory and requires power tomaintain stored information. In some embodiments, the device isnon-volatile memory and retains stored information when the digitalprocessing device is not powered. In further embodiments, thenon-volatile memory comprises flash memory. In some embodiments, thenon-volatile memory comprises dynamic random-access memory (DRAM). Insome embodiments, the non-volatile memory comprises ferroelectric randomaccess memory (FRAM). In some embodiments, the non-volatile memorycomprises phase-change random access memory (PRAM). In otherembodiments, the device is a storage device including, by way ofnon-limiting examples, CD-ROMs, DVDs, flash memory devices, magneticdisk drives, magnetic tapes drives, optical disk drives, and cloudcomputing based storage. In further embodiments, the storage and/ormemory device is a combination of devices such as those disclosedherein.

In some embodiments, the digital processing device includes a display tosend visual information to a user. In some embodiments, the display is acathode ray tube (CRT). In some embodiments, the display is a liquidcrystal display (LCD). In further embodiments, the display is a thinfilm transistor liquid crystal display (TFT-LCD). In some embodiments,the display is an organic light emitting diode (OLED) display. Invarious further embodiments, on OLED display is a passive-matrix OLED(PMOLED) or active-matrix OLED (AMOLED) display. In some embodiments,the display is a plasma display. In other embodiments, the display is avideo projector. In still further embodiments, the display is acombination of devices such as those disclosed herein.

In some embodiments, the digital processing device includes an inputdevice to receive information from a user. In some embodiments, theinput device is a keyboard. In some embodiments, the input device is apointing device including, by way of non-limiting examples, a mouse,trackball, track pad, joystick, game controller, or stylus. In someembodiments, the input device is a touch screen or a multi-touch screen.In other embodiments, the input device is a microphone to capture voiceor other sound input. In other embodiments, the input device is a videocamera or other sensor to capture motion or visual input. In furtherembodiments, the input device is a Kinect, Leap Motion, or the like. Instill further embodiments, the input device is a combination of devicessuch as those disclosed herein.

Referring to FIG. 15, in some embodiments, an exemplary digitalprocessing device 1501 is programmed or otherwise configured to loadsamples and reagents, control temperatures in the reaction vessel, coolthe reaction vessels and analyze signals from reaction vessels. Thedevice 1501 can regulate various aspects of the present disclosure, suchas, for example, increasing and decreasing the temperature of thereaction vessel using the light source(s) and the cooling channels. Inthis embodiment, the digital processing device 1501 includes a centralprocessing unit (CPU, also “processor” and “computer processor” herein)1505, which can be a single core or multi core processor, or a pluralityof processors for parallel processing. The digital processing device1501 also includes memory or memory location 1510 (e.g., random-accessmemory, read-only memory, flash memory), electronic storage unit 1515(e.g., hard disk), communication interface 1520 (e.g., network adapter)for communicating with one or more other systems, and peripheral devices1525, such as cache, other memory, data storage and/or electronicdisplay adapters. The memory 1510, storage unit 1515, interface 1520 andperipheral devices 1525 are in communication with the CPU 1505 through acommunication bus (solid lines), such as a motherboard. The storage unit1515 can be a data storage unit (or data repository) for storing data.The digital processing device 1501 can be operatively coupled to acomputer network (“network”) 1530 with the aid of the communicationinterface 1520. The network 1530 can be the Internet, an internet and/orextranet, or an intranet and/or extranet that is in communication withthe Internet. The network 1530 in some cases is a telecommunicationand/or data network. The network 1530 can include one or more computerservers, which can enable distributed computing, such as cloudcomputing. The network 1530, in some cases with the aid of the device1501, can implement a peer-to-peer network, which may enable devicescoupled to the device 1501 to behave as a client or a server.

Continuing to refer to FIG. 15, the CPU 1505 can execute a sequence ofmachine-readable instructions, which can be embodied in a program orsoftware. The instructions may be stored in a memory location, such asthe memory 1510. The instructions can be directed to the CPU 1505, whichcan subsequently program or otherwise configure the CPU 1505 toimplement methods of the present disclosure. Examples of operationsperformed by the CPU 1505 can include fetch, decode, execute, and writeback. The CPU 1505 can be part of a circuit, such as an integratedcircuit. One or more other components of the device 1501 can be includedin the circuit. In some cases, the circuit is an application specificintegrated circuit (ASIC) or a field programmable gate array (FPGA).

Continuing to refer to FIG. 15, the storage unit 1515 can store files,such as drivers, libraries and saved programs. The storage unit 1515 canstore user data, e.g., user preferences and user programs. The digitalprocessing device 1501 in some cases can include one or more additionaldata storage units that are external, such as located on a remote serverthat is in communication through an intranet or the Internet.

Continuing to refer to FIG. 15, the digital processing device 1501 cancommunicate with one or more remote computer systems through the network1530. For instance, the device 1501 can communicate with a remotecomputer system of a user. Examples of remote computer systems includepersonal computers (e.g., portable PC), slate or tablet PCs (e.g.,Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g.,Apple® iPhone, Android-enabled device, Blackberry®), or personal digitalassistants.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the digital processing device 1501, such as, for example, onthe memory 1510 or electronic storage unit 1515. The machine executableor machine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1505. In some cases, thecode can be retrieved from the storage unit 1515 and stored on thememory 1510 for ready access by the processor 1505. In some situations,the electronic storage unit 1515 can be precluded, andmachine-executable instructions are stored on memory 1510.

Non-Transitory Computer Readable Storage Medium

In some embodiments, the platforms, systems, media, and methodsdisclosed herein include one or more non-transitory computer readablestorage media encoded with a program including instructions executableby the operating system of an optionally networked digital processingdevice. In further embodiments, a computer readable storage medium is atangible component of a digital processing device. In still furtherembodiments, a computer readable storage medium is optionally removablefrom a digital processing device. In some embodiments, a computerreadable storage medium includes, by way of non-limiting examples,CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic diskdrives, magnetic tape drives, optical disk drives, cloud computingsystems and services, and the like. In some cases, the program andinstructions are permanently, substantially permanently,semi-permanently, or non-transitorily encoded on the media.

Computer Program

In some embodiments, the platforms, systems, media, and methodsdisclosed herein include at least one computer program, or use of thesame. A computer program includes a sequence of instructions, executablein the digital processing device's CPU, written to perform a specifiedtask. Computer readable instructions may be implemented as programmodules, such as functions, objects, Application Programming Interfaces(APIs), data structures, and the like, that perform particular tasks orimplement particular abstract data types. In light of the disclosureprovided herein, those of skill in the art will recognize that acomputer program may be written in various versions of variouslanguages.

The functionality of the computer readable instructions may be combinedor distributed as desired in various environments. In some embodiments,a computer program comprises one sequence of instructions. In someembodiments, a computer program comprises a plurality of sequences ofinstructions. In some embodiments, a computer program is provided fromone location. In other embodiments, a computer program is provided froma plurality of locations. In various embodiments, a computer programincludes one or more software modules. In various embodiments, acomputer program includes, in part or in whole, one or more webapplications, one or more mobile applications, one or more standaloneapplications, one or more web browser plug-ins, extensions, add-ins, oradd-ons, or combinations thereof.

Web Application

In some embodiments, a computer program includes a web application. Inlight of the disclosure provided herein, those of skill in the art willrecognize that a web application, in various embodiments, utilizes oneor more software frameworks and one or more database systems. In someembodiments, a web application is created upon a software framework suchas Microsoft® .NET or Ruby on Rails (RoR). In some embodiments, a webapplication utilizes one or more database systems including, by way ofnon-limiting examples, relational, non-relational, object oriented,associative, and XML database systems. In further embodiments, suitablerelational database systems include, by way of non-limiting examples,Microsoft® SQL Server, mySQL™, and Oracle®. Those of skill in the artwill also recognize that a web application, in various embodiments, iswritten in one or more versions of one or more languages. A webapplication may be written in one or more markup languages, presentationdefinition languages, client-side scripting languages, server-sidecoding languages, database query languages, or combinations thereof. Insome embodiments, a web application is written to some extent in amarkup language such as Hypertext Markup Language (HTML), ExtensibleHypertext Markup Language (XHTML), or eXtensible Markup Language (XML).In some embodiments, a web application is written to some extent in apresentation definition language such as Cascading Style Sheets (CSS).In some embodiments, a web application is written to some extent in aclient-side scripting language such as Asynchronous Javascript and XML(AJAX), Flash® Actionscript, Javascript, or Silverlight®. In someembodiments, a web application is written to some extent in aserver-side coding language such as Active Server Pages (ASP),ColdFusion®, Perl, Java™, JavaServer Pages (JSP), Hypertext Preprocessor(PHP), Python™, Ruby, Tcl, Smalltalk, WebDNA®, or Groovy. In someembodiments, a web application is written to some extent in a databasequery language such as Structured Query Language (SQL). In someembodiments, a web application integrates enterprise server productssuch as IBM® Lotus Domino®. In some embodiments, a web applicationincludes a media player element. In various further embodiments, a mediaplayer element utilizes one or more of many suitable multimediatechnologies including, by way of non-limiting examples, Adobe® Flash®,HTML 5, Apple® QuickTime®, Microsoft® Silverlight®, Java™, and Unity®.

Mobile Application

In some embodiments, a computer program includes a mobile applicationprovided to a mobile digital processing device. In some embodiments, themobile application is provided to a mobile digital processing device atthe time it is manufactured. In other embodiments, the mobileapplication is provided to a mobile digital processing device via thecomputer network described herein.

In view of the disclosure provided herein, a mobile application iscreated by techniques known to those of skill in the art using hardware,languages, and development environments known to the art. Those of skillin the art will recognize that mobile applications are written inseveral languages. Suitable programming languages include, by way ofnon-limiting examples, C, C++, C#, Objective-C, Java™, Javascript,Pascal, Object Pascal, Python™, Ruby, VB.NET, WML, and XHTML/HTML withor without CSS, or combinations thereof.

Suitable mobile application development environments are available fromseveral sources. Commercially available development environmentsinclude, by way of non-limiting examples, AirplaySDK, alcheMo,Appcelerator®, Celsius, Bedrock, Flash Lite, .NET Compact Framework,Rhomobile, and WorkLight Mobile Platform. Other development environmentsare available without cost including, by way of non-limiting examples,Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile devicemanufacturers distribute software developer kits including, by way ofnon-limiting examples, iPhone and iPad (iOS) SDK, Android™ SDK,BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, andWindows® Mobile SDK.

Those of skill in the art will recognize that several commercial forumsare available for distribution of mobile applications including, by wayof non-limiting examples, Apple® App Store, Google® Play, ChromeWebStore, BlackBerry® App World, App Store for Palm devices, App Catalogfor webOS, Windows® Marketplace for Mobile, Ovi Store for Nokia®devices, Samsung® Apps, and Nintendo® DSi Shop.

Standalone Application

In some embodiments, a computer program includes a standaloneapplication, which is a program that is run as an independent computerprocess, not an add-on to an existing process, e.g., not a plug-in.Those of skill in the art will recognize that standalone applicationsare often compiled. A compiler is a computer program(s) that transformssource code written in a programming language into binary object codesuch as assembly language or machine code. Suitable compiled programminglanguages include, by way of non-limiting examples, C, C++, Objective-C,COBOL, Delphi, Eiffel, Java™, Lisp, Python™, Visual Basic, and VB .NET,or combinations thereof. Compilation is often performed, at least inpart, to create an executable program. In some embodiments, a computerprogram includes one or more executable complied applications.

Web Browser Plug-In

In some embodiments, the computer program includes a web browser plug-in(e.g., extension, etc.). In computing, a plug-in is one or more softwarecomponents that add specific functionality to a larger softwareapplication. Makers of software applications support plug-ins to enablethird-party developers to create abilities which extend an application,to support easily adding new features, and to reduce the size of anapplication. When supported, plug-ins enable customizing thefunctionality of a software application. For example, plug-ins arecommonly used in web browsers to play video, generate interactivity,scan for viruses, and display particular file types. Those of skill inthe art will be familiar with several web browser plug-ins including,Adobe® Flash® Player, Microsoft® Silverlight®, and Apple® QuickTime®. Insome embodiments, the toolbar comprises one or more web browserextensions, add-ins, or add-ons. In some embodiments, the toolbarcomprises one or more explorer bars, tool bands, or desk bands.

In view of the disclosure provided herein, those of skill in the artwill recognize that several plug-in frameworks are available that enabledevelopment of plug-ins in various programming languages, including, byway of non-limiting examples, C++, Delphi, Java™, PHP, Python™, and VB.NET, or combinations thereof.

Web browsers (also called Internet browsers) are software applications,designed for use with network-connected digital processing devices, forretrieving, presenting, and traversing information resources on theWorld Wide Web. Suitable web browsers include, by way of non-limitingexamples, Microsoft® Internet Explorer®, Mozilla® Firefox®, Google®Chrome, Apple® Safari®, Opera Software® Opera®, and KDE Konqueror. Insome embodiments, the web browser is a mobile web browser. Mobile webbrowsers (also called mircrobrowsers, mini-browsers, and wirelessbrowsers) are designed for use on mobile digital processing devicesincluding, by way of non-limiting examples, handheld computers, tabletcomputers, netbook computers, subnotebook computers, smartphones, musicplayers, personal digital assistants (PDAs), and handheld video gamesystems. Suitable mobile web browsers include, by way of non-limitingexamples, Google® Android® browser, RIM BlackBerry® Browser, Apple®Safari®, Palm® Blazer, Palm® WebOS® Browser, Mozilla® Firefox® formobile, Microsoft® Internet Explorer® Mobile, Amazon® Kindle® Basic Web,Nokia® Browser, Opera Software® Opera® Mobile, and Sony® PSP™ browser.

Software Modules

In some embodiments, the platforms, systems, media, and methodsdisclosed herein include software, server, and/or database modules, oruse of the same. In view of the disclosure provided herein, softwaremodules are created by techniques known to those of skill in the artusing machines, software, and languages known to the art. The softwaremodules disclosed herein are implemented in a multitude of ways. Invarious embodiments, a software module comprises a file, a section ofcode, a programming object, a programming structure, or combinationsthereof. In further various embodiments, a software module comprises aplurality of files, a plurality of sections of code, a plurality ofprogramming objects, a plurality of programming structures, orcombinations thereof. In various embodiments, the one or more softwaremodules comprise, by way of non-limiting examples, a web application, amobile application, and a standalone application. In some embodiments,software modules are in one computer program or application. In otherembodiments, software modules are in more than one computer program orapplication. In some embodiments, software modules are hosted on onemachine. In other embodiments, software modules are hosted on more thanone machine. In further embodiments, software modules are hosted oncloud computing platforms. In some embodiments, software modules arehosted on one or more machines in one location. In other embodiments,software modules are hosted on one or more machines in more than onelocation.

Databases

In some embodiments, the platforms, systems, media, and methodsdisclosed herein include one or more databases, or use of the same. Inview of the disclosure provided herein, those of skill in the art willrecognize that many databases are suitable for storage and retrieval ofinformation such as protocols, cycle times, temperature ranges, results,detection results and reports. In various embodiments, suitabledatabases include, by way of non-limiting examples, relationaldatabases, non-relational databases, object oriented databases, objectdatabases, entity-relationship model databases, associative databases,and XML databases. Further non-limiting examples include SQL,PostgreSQL, MySQL, Oracle, DB2, and Sybase. In some embodiments, adatabase is internet-based. In further embodiments, a database isweb-based. In still further embodiments, a database is cloudcomputing-based. In other embodiments, a database is based on one ormore local computer storage devices.

Referring to FIG. 16, a method 1600 for determining a target nucleicacid modification is shown. The method 1600 may use one or more of thesystems described herein. In a first step 1610, samples and/or reagentsrequired for the modification of nucleic acids may be loaded on thereaction vessel or reaction wells. In some cases, the reagents may bepre-loaded on to the wells of the reaction vessel. In a second step1620, the reaction vessel may be placed on the transparent block. Insome cases, the reaction vessel may be placed above the base with thelight supports without the use of an additional transparent block. Thereaction vessel may be sealed using a sealing film or a sealing platedescribed herein. In a third step 1630, the reaction vessel may beheated using a light source. The light source may be operated in acontinuous or in a pulsed manner as described herein. The fourth step1640, the reaction vessel may be cooled using a fluid circulationchannel in the transparent block. In a fifth step 1650, an excitationsource may be used to excite the fluorescent dyes integrated during thenucleic acid modification process. This step may be performed during thenucleic acid modification cycles or after the nucleic acid modificationcycles. In a sixth step 1660, sensors may be used to detect therefracted light from the wells. In a seventh step 1670, the signals fromthe plurality of reaction wells may be reported as an output.

In some instances, a processor may be provided. The processor may beconfigured with instructions to perform a series of steps illustrated inFIG. 16 and others as described herein. In some instances, the processormay provide instructions for the modification of nucleic acids anddetection of a target nucleic acid. A processor may be used to performsome protocols for nucleic acid modification, to pulse a light source oran excitation source. A processor may also be used to cool the reactionvessel at different time intervals.

Although the steps described above show a method of modification ofnucleic acids and detection of a target nucleic acid, one of ordinaryskill in the art will recognize many variations based on the teachingsdescribed herein. The steps may be completed in a different order. Stepsmay be added or deleted. Some of the steps may comprise sub-steps. Manyof the steps may be repeated as often as necessary to detect the nucleicacid as desired. In some embodiments, a processor is configured toperform one or more steps of a method as described herein.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

EXAMPLES Example 1: Use as a Point of Care Device

Due to the high co-infection rate of Chlamydia trachomatis (CT) andNeisseria gonorrhea (NG) and the ability of NG to mutate to a variety ofgonococcal antimicrobial-resistant (AMR) strains, high-throughputdetection is beneficial for diagnosing CT/NG and NG AMR. Therefore,high-throughput multiplexed PCR-based point of care testing would bebeneficial. Presented in FIG. 17 is a systematic diagnostic solution fordetecting CT/NG in 15 min and follow-up AMR test for NG upon positivetest result in additional 15 min, delivering a fully reliable diagnosticdecision within the single test. This systematic approach mayinclude; 1) a cartridge-based sample preparation module; 2) a pneumaticpressure driven liquid handling platform; 3) a photonic PCR thermalcycler; and 4) a microfluidic array device as described previouslyherein for high-throughput DNA amplifications of CT/NG and gonococcalAMR. Positive results of these tests may help in generating an effectivetreatment plan.

Example 2: Representative End-Point Photonic PCR Data for Various GeneSequences

FIGS. 18A-C shows the demonstration of the end-point photonic PCR forvarious amplifications, including DENV-1 cDNA (dengue fever), crtA (forN. meningitidis), hpd (for Haemophilus influenzae), and mecA (forMethicillin-resistant S. aureus). Purified nucleic acids (RNA for denguefever converted to cDNA and DNA for crtA, hpd and mecA) were used fordetection. Two-step PCR thermal cycling from 98° C. to 68° C. wasemployed with 40 cycles of amplification. Reaction volume of PCR was 10μL. The amplified product was then run on a gel and compared to resultsfrom conventional PCR used as a positive control. The gel band intensityshows a clear trend as a function of template DNA concentration (FIGS.18A and 18C). Multiplex photonic PCR is also demonstrated using crtA andhpd multiplexed reactions (FIG. 18B). It is noted that as low as 5copies of mecA gene and 10³ viral particles per ml (vp/ml) weresuccessfully amplified and confirmed by gel electrophoresis afterphotonic PCR amplification (FIGS. 18A and 18C).

What is claimed is:
 1. A system comprising: a polymeric fluidic devicecomprising one or more reaction wells; a first support comprising aconcave region; a first light absorbing material disposed on the concaveregion of the first support; a second support opposite the firstsupport, wherein the second support comprises a convex region, whereinthe first light absorbing material and the convex region of the secondsupport define a reaction well of the one or more reaction wells; firstand second ports coupled to the one or more reaction wells; wherein thefirst and second ports are configured to allow input of a fluidic sampleinto the one or more reaction wells; a lyophilized reagent is pre-loadedon the one or more reaction wells; and a light source configured toilluminate the first light absorbing material; wherein a first portionof light illuminated onto the first light absorbing material is absorbedinto the first light absorbing material; and wherein the first portionof light absorbed into the first light absorbing material is configuredto elevate a temperature of the first light absorbing material to heatthe fluidic sample within the one or more reaction wells.
 2. The systemof claim 1 wherein: a second light absorbing material is disposed on theconvex region of the second support; a second portion of the lightilluminated onto the first light absorbing material is transmittedthrough the first light absorbing material and illuminates the secondlight absorbing material; and at least a portion of the transmittedlight illuminated onto the second light absorbing material is absorbedinto the second light absorbing material; and the light absorbed intothe second light absorbing material is configured to elevate atemperature of the second light absorbing material to further heat thefluidic sample within the one or more reaction wells.
 3. The system ofclaim 1 further comprising a first filter for emission of fluorescentdye and a second filter for elimination of light from the light source.4. The system of claim 1 wherein the lyophilized reagent furthercomprises a stabilizing reagent including paraffin wax or hydrogel. 5.The system of claim 1 further comprising a fluidic valve between wells.6. The system of claim 1 wherein the polymeric fluidic device comprisesa fluid circulation channel.
 7. The system of claim 6 wherein at leastone of air, water, or a liquid flows through the fluid circulationchannel.
 8. The system of claim 1 wherein the light source comprises apulsed light source.
 9. The system of claim 8 further comprising adetector operable to detect a signal indicating nucleic acidsmodification during an off cycle of the pulsed light source.
 10. Thesystem of claim 1 wherein the first light absorbing material comprisesone or multiple open areas.
 11. A system comprising: a samplepreparation module comprising multiple compartments and a cartridge,wherein the multiple compartments comprises a sample compartment, one ormore wash buffer compartments, an elution buffer compartment, and awaste compartment; wherein the cartridge comprises: a first microfluidicchannel layer comprising a passageway connected to the samplecompartment a second microfluidic channel layer comprising a firstreservoir in communication with the passageway in the first microfluidicchannel layer, wherein the second microfluidic channel layer comprises:a collection area; and a first microfluidic channel connecting the firstreservoir to the collection area; and a microfluidic PCR deviceincluding photonic PCR wells in fluid communication with the collectionarea via a second microfluidic channel.
 12. The system of claim 11wherein the sample preparation module comprises one or more filters in acompartment at least one of the multiple compartments.
 13. The system ofclaim 12 wherein the one or more filters comprise a first filter and asecond filter, wherein the first filter and the second filter havedifferent pore sizes.
 14. The system of claim 13 wherein: the firstfilter is configured to remove large debris, crystals and/or large cellsfrom a sample; and the second filter is configured to trap cells ofinterest based on the size of cells.
 15. The system of claim 13 whereinthe second filter comprises a layer of light absorbing material.
 16. Thesystem of claim 15 wherein the layer of light absorbing material isdisposed in at least one of the multiple compartments below the secondfilter.
 17. The system of claim 11 wherein the sample compartmentcomprises electrodes placed around the sample compartment to change thepH of a solution disposed in the sample compartment by applying voltage.18. The system of claim 11 wherein the waste compartment comprises atleast one of absorbing porous paper, fabric, or a sponge to preventre-fluxes of fluid.
 19. The system of claim 11 wherein the microfluidicPCR device includes a set of primers and probes to detect one or moretarget nucleic acids.
 20. The system of claim 11 wherein the systemcomprises a light absorbing material including one or more open areas.21. The system of claim 11, further comprising: a second reservoir influid communication with the one or more wash buffer compartments; athird microfluidic channel connecting the second reservoir to thecollection area; a third reservoir in fluid communication with theelution buffer compartment; and a fourth microfluidic channel connectingthe third reservoir to the collection area.
 22. The system of claim 11wherein the collection area comprises a light absorbing material. 23.The system of claim 11 wherein the collection area comprises a filterbetween the first microfluidic channel layer and the second microfluidicchannel layer.